��Universal Journal of Plant Science 1(4): 113-117, 2013 http://www.hrpub.org DOI: 10.13189/ujps.2013.010401 Cytotoxic Activities of Extracts of Medicinal Plants of Euphorbiacae Family Studied on Seven Human Cancer Cell lines Ekta Prakash*, D.K.Gupta Department of Biochemistry, Allahabad University, India *Corresponding author: [e-mail] Copyright � 2013 Horizon Research Publishing All rights reserved. Abstract Plant extracts of species of the family 1996).There seems to be increasing possibility of finding Euphorbiaceae used by traditional healers for the treatment biological activity among plants with recorded medicinal of ulcers, cancers, tumors, warts and other diseases. were uses rather than from plants randomly selected (Unander et tested in vitro for their potential anti-proliferative activity. al. 1995, Cordell 1995). Furthermore, selection of plants The objective of the present study was to evaluate the gives better criteria for screening programs especially in its in-vitro anti- cancer effects of ethanolic extract of three plant initial phases, compared to the screening of compounds species namely Ricinus communis Linn, Euphorbia isolated or purified from natural products (Kusumoto et al. helioscopia, Jatropha curcas of the family Euphorbiaceae 1995, Cordell 1995, Baker et al. 1995). The objective of our by SRB assay against seven human cancer cell lines. Colon work was to evaluate using SRB assay, the in vitro cytotoxic cancer cell line (Colon HT-29, SW-20, SiHa, Colon 502717), activity of some Euphorbia species that are known in India to Liver cancer cell line (Hep-2), Breast cancer cell line have traditional medicinal uses against cancers. (T-47D), Cervix cancer cell line OVCAR-5, Prostrate cancer cell line (PC-3) and Lungs (AF-49). The SRB assay was done in replicates to test cytotoxic activity of the three above 2. Materials and Methods mentioned plants against seven human cancer cell lines. The activity was evaluated at 100 �g/ml concentration of test 2.1. General Method of Ethanolic Extract Preparation of material. Jatropa curcas showed 47% activity against SiHa. Three Plants The ethanolic fraction of seed part of Ricinus communis showed 41% activity against Colon 502713 while stem part The plant part was placed in glass percolator of exhibited maximum activity against SiHa (47%). The appropriate size. Sufficient quantity of solvent was added to ethanolic extract of Euphorbia helioscopia inhibited the submerge the plant material. After standing for about 16 growth of three cancer cell lines viz Hep-2, T-47D and hours percolate was collected and filtered if required. The PC-5.Hep-2 showed 27% activity. process was repeated four times for exhaustive extraction of the plant material. The ethanolic extract was evaporated Keywords Euphorbiaceae, Ricinus Communis, Jatropha, under reduce pressure at 50oC using rotavapor and round E.Helioscopia, Cytotoxic Activity bottom flask. The finally it was concentrated in a vacuum desiccators. The extract was transferred to glass container of appropriate size. This form the stock extract. 2.1.1. Source of Human Cancer Cell Line 1. Introduction Human cancer cell line were obtained from National Several plants of Euphorbiaceae family have been tested Centre for cell science, Pune 411007 (India) and National for their anticancer property, partly based on information Cancer Institute, DTCD, Fredrick Cancer Research and concerning plants that have traditionally been used as Development Centre, Fairview centre, Suite 205, 1003, West medication to treat various human diseases (Bernal &Correa -7 th Street Frederick MD 21701- 8527 (USA) 1990, Unander et al.1995). Antitumor activity against 2.1.2. Selection of Human Cancer Cell Line sarcoma 180 ascites, leukemia in mice and cytotoxic activity The cell line were selected in such a way that almost all the against certain cancer cell lines has also been observed cell line grow on a single growth medium (RPMI-1640) in (Itokawa et al. 1989, Wu et al. 1991, Fatope et al. 114 Cytotoxic Activities of Extracts of Medicinal Plants of Euphorbiacae Family Studied on Seven Human Cancer Cell lines tissue culture flask (TCP) and the mass doubling time was SRB assay was carried out as described by Skehan et al., such that enough cell were obtained for screening. Cell 1990, using SRB dye in replicates. After 48 hours incubation which were used were free from bacteria, yeast, mould, of cells with test material, the plates were taken out and 50 �l mycoplasma and in special cases from viruses at all the of chilled 50% TCA was gently layered on top of the stages. If contamination appeared at any stage, the stock in medium in all the wells to produce a final concentration of which it occurred was discarded immediately. Cancer of 10%. After that Tissue culture plate were incubated at 4 �C central nervous system CNS, Lung cancer cell line A-549, in a refrigerator to fix the cells attached to the bottom of the Colon cancer cell lines, Colo-205, Colon 502713, Liver wells. After one hour the plates were taken out from cancer cell line, Hep-2, Ovarian cancer cell line, OVCAR-5, refrigerator and all the contents of all the wells were pipetted Prostrate cancer cell line PC-5 were taken for the study. out and supernatant was discarded. The plates were washed five times with distilled water to remove TCA growth medium, low molecular metabolites, serum protein etc. For 2.2. Procedure for In Vitro Cytotoxicity Assay of Plants washing, the wells of Tissue culture plates were filled with Extract distilled water and the liquid in the wells was discarded by Cytoxicity of test sample was performed against seven sharply flicking plate over sink. Plates were air dried and can human cancer cell lines in replicates. 96 well flat bottom be stored until use. SRB solution (100 �l) was added to each tissue culture plates were taken. There were four types of well of the plates and the plates were incubated for 30 well in TCP, control blank (CB, without cells, complete minutes at room temperature. The unbound SRB was growth medium only) and control growth (GC, with cell in removed quickly (to avoid desorption of protein bound dye) absence of test material) to determine 100% growth. The by washing the wells of the plates five times with 1 % acetic growth in the presence of test material was determined from acid. Plates were than air dried. After that Tris buffer (100 �l the difference of test growth (GT, cell with test material) and /well) was added in the plates. The plates were gently stirred test control (CT, test material without cells). The desired for 5 minutes on a mechanical shaker and optical density was human cancer cell lines were grown in tissue culture flask at recorded on ELISA reader at 540 nm. 37oC in an atmosphere of 5% in CO2 and 90% relative humidity in complete growth medium to obtain enough number of cells. The cells were harvested by the treatment of 3. Results and Discussion trypsin  EDTA and complete growth medium added. Viable The aim of this study was to evaluate the anti-cancer cells were counted in haemocytometer by using trypan blue. activity of three Euphorbiacae plants namely Ricinus Viable cell density was adjusted 5000- 40,000 cells/100 �l communis, Euphorbia helioscopia and Jatropa curcas. The depending upon the cell line (Monks et al 1991). Cell cytotoxic activity of these plants was determined against suspension 100 �l was added. Complete growth medium was seven human cancer cell lines. Following seven human added and incubated at 37oC for 24 hours in an atmosphere cancer cell lines were taken Colon cancer cell line (Colon of 5% CO2 and 90% relative humidity in a CO2 incubator. HT-29, SW-20, SiHa), Liver cancer cell line (Hep-2), Breast After 24 hours test material was added. Plates were cancer cell line (T-47D), Cervix cancer cell line OVCAR-5, incubated at 37�C for 48 hours in an atmosphere of 5% CO2 Prostrate cancer cell line (PC-3). SRB assay was done in and 90% relative humidity in a CO2 incubator. The growth replicates to determine the cytotoxic activity of these plants. was determined after 48 hours by SRB assay. The results summarized in Table1.shows anti-proliferative activity of three plants., 2.3. SRB Assay Table1. In- Vitro Cytotoxicity of Plant Extract of Euphorbia helioscopia, Ricinus communis Linn and Jatropha curcas Against Human Cancer Cell Line Ovary Plant Conc Breast Colon Prostrate SW- Colon Plant name Hep-2 OVCA part �g/ml T-47D HT-29 PC-3 620 SiHa R-5 % Growth Inhibition Whole 1. Euphorbia helioscopia 100 27 7 0 11 -- - - Plant 2. Ricinus communis Linn. 100 9 - 31 - 0 40 - Stem 3. Jatropha curcas Leaves 100 - 0 - - - 47 30 (-) means that extract was not evaluated with particular human cancer cell line  Universal Journal of Plant Science 1(4): 113-117, 2013 115 Figure 1. The ethanolic extract of Ricinus communis inhibited the growth of only four cancer cell lines viz colon 502713, A-549, OVCAR-5 and PC-5.The cytotoxic activity was observed in Colon 502713, A549, OVCAR-5, PC-5 are 41%, 11%, 12%, 14% respectively. Extract was observed to show no cytototoxic activity against these human cancer cell lines SF-295, Colo-205, Hep-2. In vitro cytotoxicity of Euphorbia helioscopia (ethanolic extract) against human cancer cell lines Figure 2. The ethanolic extract of Euphorbia helioscopia inhibited the growth of only three cancer cell lines viz Hep-2 (27%), T-47D (7%) and PC-5(11%). The cytotoxic activity was observed to be nil against following cancer cell lines SW-670, HCT-15, SiHa, OVCAR-5. In vitro cytotoxicity of Jatropa curcas (ethanolic extract) against human cancer cell lines 116 Cytotoxic Activities of Extracts of Medicinal Plants of Euphorbiacae Family Studied on Seven Human Cancer Cell lines Table 2. In-Vitro Cytotoxicity of Plant Extract of Ricinus communis Linn (seed part) Against Human Cancer Cell Lines Plant Conc Colon Colon Liver Lung Ovary Prostrate Plant name SF295 part �g/ml 502713 Colo205 Hep-2 A-549 Ovcar-5 PC-5 % Growth Inhibition 1. Ricinus communis Linn. Seed 100 - 41 - - 11 12 14 (-) means that extract was not evaluated with particular human cancer cell line Figure 3. The ethanolic extract of Jatropa curcas showed nil activity against T-47D Human cancer cell line. HCT-15, SiHA, OVCAR-5 showed 47 %, 13 % and 14 % activity. Total seven human cancer cell lines were taken to study In conclusion. the highly potent activities exhibited by the extract of seed and stem part of Ricinus communis. The Jatropa curcas and Ricinus communis and Euphorbia ethanolic extract of seed part of Ricinus communis found helioscopia even at low concentration (100�g/ml) suggest active against Colon 502713, A-549, OVCAR-5 and PC-5 that these compounds could be developed further as human cancer cell lines. The cytotoxic activity found to be anticancer drugs. 41%, 11%, 12% and 14% against Colon 502713, A549, OVCAR-5, PC-5 respectively. The ethanolic extract of stem part of Ricinus communis shows 9%, 31% and 40% activity Acknowledgements against Hep-2, HT-29 and SiHa cell lines at 100 �g/ml I would like to thank senior scientist Dr.Ajit.K Saxena, concentration. Fig1.shows cytotoxic activities of Ricinus Indian Institute of Integrative Medicine (C.S.I.R Lab), Tawi, communis against mentioned cell lines. The ethanolic extract Jammu, India for providing us human cancer cell lines and of Euphorbia helioscopia inhibited the growth of three giving infrastructural facility to perform my experiments human cancer cell lines namely Hep-2, T-47D and PC-5. without which such study on anticancer plant was not 27%, 7% and 11% activity was observed against these three possible. cell lines respectively. Figs 2 explain the percentage activity against human cancer cell lines. The cytotoxic activity of ethanolic fraction of Jatropa curcas was found to be 47 % and 30% against SiHa and OVCAR-5 human cancer cell lines respectively as shown in Table 2. REFERENCES  Universal Journal of Plant Science 1(4): 113-117, 2013 117 [1] Ahmad, B., Alam, T., Varshney, M. and Khan, S. A. (2002) nervous system stimulant isolated from Ricinus communis. Hepatoprotective activity of two plants belonging to the Pharmacol Biochem Be. 1999; 63:367 375. doi: Apiaceae and the Eurphorbiaceae family.J Ethnopharmacol. 10.1016/S0091-3057(99)00007-6 79, p. 313. [12] Kirtikar KR, Basu BA. Indian Medicinal Plants. 3. 1991. pp. [2] Ahmad, I., Mehmood, Z. and Mohammad, F. (1998) 2274 2277. Screening of some Indian medicinal plants for their [13] Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN, Ray C. antimicrobial properties. J Ethnopharmacol.62, p. 183. Screening of Indian plants for biological activity. Part I. [3] Ahmad, I., Mahmood, M., Mohammad, F. and Ahmad, S. Indian J Exp Biol. 1998; 6:232 247. [PubMed] (2001) Antimicrobial potency and synergistic activity of five [14] Capasso F, Mascolo N, Izzo AA, Gaginella TS. Dissociation traditionally used Indian medicinal plants.J Med and Aromat of castor oil induced diarrhea and intestinal mucosal injury in Plant Sci. 23, p. 173. rat: effect of NG-nitro-L- arginine methyl ester. B J [4] Bravo.L. (1998) Polyphenols: Chemistry, dietary sources, Pharmacol. 1994; 113:1127 1130. doi: metabolism, and nutritional significance. Nutr Rev 10.1111/j.1476-5381.1994.tb17113.x.] 56:317-333 [15] Jimu L, Nyakudya IW, Katsvanga CAT. Establishment and [5] Bani S, Kaul A, Jaggi BS, Suri KA, Suri OP, Sharma early field performance of Jatropha curcas L at Bindura OP.(2000) Anti-inflammatory activity of the hydrosoluble University Farm, Zimbabwe. Journal of Sustainable fraction of Euphorbia royleana latex. Fitoterapia; Development in Africa. 2009; 10:445 469. 71(6):655-62. [16] Von Linn� C. Species Plantarum. 1st edition. Stockholm, [6] Betancur-Galvis LA, Saez J, Granados H, Salazar A, Ossa JE Sweden: Laurenti Salvi; 1753. (1999) Antitumor and antiviral activity of Colombian [17] Von Linn� C. Systema naturae, sive regna tria natur� medicinal plant extracts. Mem Inst Oswaldo Cruz systematice proposita per classes, ordines, genera, species. 94:531-535. Tomus II; Pars II. Lipsiae, Germany: J. F. Gmelin; 1791. [7] Fatope MO, Zeng L, Ohayaga JE, Shi G, McLaughlin [18] De Jussieu AL. Genera Plantarum Secundum Ordines JL(1996) Selectively cytotoxic diterpenes from Euphorbia Naturales Deposit: juxta methodum in Horto Regio Parisiensi poisonii Med Chem; 39(4):1005-8. Exaratam. Herissant: Parisiis; 1789. [8] Falsone G, Crea AE, Noack EA (1982) Constituents of [19] Thomas OO. Re-examination of the antimicrobial activities Euphorbiaceae, 7. 20- Deoxyingenol Monoesters and ingenol of Xylopia aethiopica, Carica papaya, Ocimum gratissimum diesters from Euphorbia biglandulosa Desf. Arch Pharm and Jatropha curcas. Fitoterapia. 1989;60(2):147 155. (Weinheim) 12: 1026-1032. [20] Ekta Prakash.Analytics of Medicinal and Latex bearing plants [9] Denizot F, Lang R 1986. Rapid colorimetric assay for cell for possible anticancer and antiarthritic properties using grow and survival modifications to the tetrazolium dye in-vitro studies:2013 procedure giving improved sensitivity and reliability. J Immunol Meth 89: 271-277 [21] Burkill HM. The Useful Plants of West Tropical Africa. Vol. 2. Kew, UK: Royal Botanical Gardens; 1994. [10] Fatope MO, Zeng L, Ohayaga JE, Shi G, McLaughlin JL 1996.Selectively Cytotoxic diterpenes from Euphorbia [22] Hirata Y. Toxic substances of Euphorbiaceae. Pure and poisonii J Med Chem 39: 1005-1008. Applied Chemistry. 1975; 41:175 199. [11] Ferraz A. Pharmacological evaluation of ricinine, a central